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目的:探究心脏心肌细胞与成纤维细胞间通过远距离连接——膜纳米管直接传输线粒体的生理学意义以及具体的传输机制。方法:通过激光共聚焦仪器的活细胞追踪功能,实时观测线粒体的运动及传输;利用免疫荧光双染的方法,观察线粒体与微管、微丝等结构的共定位,并使用微管抑制剂Nocodazole,阻断微管的存在,以观察微管对线粒体传输的作用;使用Western blot、real-time PCR方法检测KIF5B蛋白的表达,继而通过使用转染KIF-5B-si RNA慢病毒方法敲减细胞中KIF5B,探究KIF5B的作用;运用TUNEL染色及高内涵检测的方法,检测心肌细胞的凋亡。结果:原代乳大鼠心肌细胞与成纤维细胞间形成的膜纳米管可以传输线粒体,线粒体在其中的平均运动速度(17.5±2.1)nm/s。在膜纳米管中,线粒体与微管存在共定位,线粒体的传输依赖于微管,并且KIF5B是膜纳米管中推动线粒体传输的马达蛋白,使线粒体可以从成纤维细胞中传输到心肌细胞中。在心肌细胞的缺氧-复氧模型的病理模型中,成纤维细胞通过将自身的线粒体传输到心肌细胞中,起到减少心肌细胞凋亡的保护作用。结论:作为一种新型的细胞连接,心肌细胞与成纤维细胞间形成的膜纳米管可以通过直接传输线粒体,从而减少受损心肌细胞的凋亡,这种传输依赖于微管及马达蛋白KIF5B。
OBJECTIVE: To investigate the physiological significance of mitochondria and its specific transport mechanism by direct connection between cardiac myocytes and fibroblasts via long-distance membrane-membrane nanotubes. Methods: The mitochondrial motility and transmission were observed in real time by laser confocal laser tracing. The co-localization of mitochondria, microtubules and microfilaments was observed by immunofluorescence double staining. The microtubule inhibitor Nocodazole , And blocked the presence of microtubules to observe the effect of microtubules on mitochondrial transport. The expression of KIF5B protein was detected by Western blot and real-time PCR, and then knocked down by using the lentivirus transfected with KIF-5B-si RNA In KIF5B to explore the role of KIF5B; use of TUNEL staining and high content of detection methods to detect myocardial apoptosis. RESULTS: Membrane nanotubes formed between primary myocytes and fibroblasts could transmit mitochondria with an average velocity of 17.5 ± 2.1 nm / s. In membrane nanotubes, mitochondria and microtubules exist co-localization, mitochondrial transport is dependent on microtubules, and KIF5B is a motor protein that drives mitochondrial transport in membrane nanotubes, allowing mitochondria to be transported from fibroblasts to cardiomyocytes. In the hypoxic-reoxygenation model of cardiomyocytes, fibroblasts play a protective role in reducing cardiomyocyte apoptosis by transmitting their mitochondria to cardiomyocytes. CONCLUSION: As a novel cell junction, membrane-derived nanotubes formed between cardiomyocytes and fibroblasts can reduce the apoptosis of damaged cardiomyocytes through the direct transport of mitochondria, which is dependent on microtubules and motor protein KIF5B.